Cosmetic use of phytosphingosine as slimming agent and cosmetic compositions comprising phytosphingosine

ABSTRACT

The invention relates to novel cosmetic uses of phytosphingosine or of one of its cosmetically acceptable salts, particularly its hydrochloride, as a slimming agent and/or as an active agent which stimulates the synthesis of leptin by adipocytes, for preparing a cosmetic composition intended for reducing subcutaneous excess fat. The invention also relates to a method of cosmetic treatment intended for obtaining a slimming effect on the human body according to which a cosmetic composition containing phytosphingosine or one of its cosmetically acceptable salts, particularly its hydrochloride, is applied on the parts of the body to be treated. The invention also relates to novel cosmetic compositions containing phytosphingosine or one of its cosmetically acceptable salts, particularly its hydrochloride, in combination with a lipolytic agent selected from the group consisting of CAMP and its derivatives, adenylate cyclase enzyme activating agents and phosphodiesterase enzyme inhibiting agents.

The present invention relates to a novel cosmetic use ofphytosphingosine, as a slimming agent, as well as to cosmeticcompositions containing phytosphingosine.

Phytosphingosine is of the following formula:

Its molecular formula is C₁₈H₃₉NO₃ and its CAS number is as follows: RN100 000 403-19-8.

This product is also known under the designation(2S,3S,4R)-2-amino-1,3,4-octadecanetriol.

Phytosphingosine is a commercial product which corresponds to one of thethree sphingoid bases which are present naturally in the skin,phytosphingosine being present in the stratum corneum.

Applications of phytosphingosine and of its salts, and, moreparticularly, of its hydrochloride, are already known in the field ofdermatology. Phytosphingosine is in fact known essentially for itsanti-microbial activity, as well as for its activity as a <<secondmessenger>>, which is an application which results in a reduction of thesensitivity of the skin. More specifically, phytosphingosine is knownfor its activity in the treatment of acne, for its activity ofinhibiting the growth of microorganisms on the skin, and for reducingvarious inflammatory phenomena observed on the skin.

The inventors of the present invention have, in an entirely surprisingway, now discovered a novel use of phytosphingosine, as well as of itscosmetically acceptable salts, particularly of its hydrochloride, as aslimming agent. They have furthermore demonstrated that this novel useis linked, at least partially, to the perfectly unexpected property ofphytosphingosine, and of its salts, of promoting the synthesis of leptinby adipocytes of the skin.

Furthermore, in pursuing their studies in this field, the inventors ofthe present invention have also demonstrated that a certain number ofcombinations of phytosphingosine or of its cosmetically acceptable saltsturned out to be particularly interesting in these novel uses.

Leptin from the mouse has recently been identified in 1994 as being theproduct of the “ob gene” (Zhang, y et al., Nature, 1994, 372:425 andTartaglia L. A., 1997, J. Biol. Chem. 272:6093).

The structure of human leptin (or human OB protein) and its use in themodulation of weight in animals are described in British patentapplication GB 2,292,382. Leptin is a protein which is secreted by theadipocyte which informs the brain of the state of the adipose reserves.It acts through membrane receptors which are situated in particular inthe hypothalamus.

Leptin was first studied in the rodent and then in man, and plays a keyrole in the regulation of body weight.

In ob/ob mice, the absence of leptin in the serum, due to mutations ofthe ob gene (that which encodes leptin), leads to a massive obesity.

In man, the first pieces of work in relation to leptin were directedtowards obese and/or diabetic patients.

In fact, the more an adipocyte possesses a higher content oftriglycerides, the more it produces leptin, and vice versa(Medecine/Sciences, 1998, no8-9, 14, 858-864, G. Ailhaud: L'adipocyte,cellule secrétrice and endocrine (<<The adipocyte, a secretory andendocrine cell>>)).

Thus, in the obese person, two situations can arise. Either a mutationof the leptin gene exists, this mutation is then non-functional,particularly on the receptors in the brain, or, a lack of transfer ofleptin exists about the blood-brain barrier.

One study, during which a daily injection of synthetic leptin was madein patients suffering from obesity or from excess weight, has shownconclusive results: a significant weight loss appeared in patientssuffering from a certain form of obesity (work carried out at TuftsUniversity in the USA, presented on the occasion of the conference:American Diabetes Association: Jean Mayer, USA Human Nutrition ResearchCenter on Aging).

Leptin does in fact trigger off a phenomenon of satiety which causes areduction in food ingestion and which reduces the frequency of foodingestion.

When the adipose mass increases, the leptin produced by the adiposetissue will inhibit the food ingestion and will stimulate energyexpenditure. Leptin will thus act against an excessive weight gain.

Hence, it may be considered that this protein is a regulator of theadipose mass, the prime role of which is to inhibit the deposit ofexcess adiposity.

The role of local regulation which is played by leptin is well-known(see Systemically and Topically Administered Leptin Both AccelerateWound Healing in Diabetic ob/ob Mice. B. D. Ring, S. Scully, C. R.Davis, M. B Baker, M. J. Cullen, M. A. Pelleymounter, D. Danilenko.Endocrinology, 2000 vol. 141, no1, p. 446-449).

Furthermore, the role of leptin in the expression of certain genesleading to the accumulation of lipids (differentiation genes) is alsowell-known within the context of regulation of lipolysis.

Leptin on the one hand supresses the expression of certain genes leadingto an accumulation of lipids (differentiation genes), and this takesplace without the participation of the brain.

On the other hand, leptin induces a lipolysis directly on theadipocytes. This has been observed on mouse adipocytes in vitro (see Invitro Lipolytic Effect of Leptin on Mouse Adipocytes: Evidence for apossible Autocrine/Paracrine Role of Leptin, G. Frühbeck, M. Aguado, J.A. Martinez, Biochemical and Biophysical Research communications 1997:240, p. 590-594). The effect of leptin on the lipolysis of theadipocytes is specific and operates via receptors which are present inthe white adipose tissue.

Leptin converts oestrone of the blood circulation (a hormone whichincreases lipid deposits) into oleyl-oestrone which is considered to bea “slimming” factor. The appearance of this factor causes a generalisedlipolysis and a thermogenesis (see Leptin enhances the synthesis ofoleyl-estrone from estrone in white adipose tissue, M. Esteve, J.Virgili, H. Aguilar, F. Balada, J. A. Fernandez-Lopez, W. Remesar, M.Alemany, Eur J. Nutr. 1999, 38, p. 99-104). When oleyl-oestrone isadministered to obese or normal rats, it causes a loss in fatty mass.

All the interest that there is is thus seen in having a means availableto act upon the synthesis of leptin, which will notably act

-   -   directly on the skin via receptors present therein,    -   on the adipose tissue in causing lipolysis,    -   on a factor acting as a weight regulator, namely,        oleyl-oestrone.

Tests carried out within the context of the present invention, oncultures of murine adipocytes as well as on cultures of humanadipocytes, have enabled demonstrating that it was possible, by treatingthese cultures with phytosphingosine or one of its salts, particularlyits hydrochloride, to stimulate the synthesis of leptin by theseadipocytes, and this left predicting the possibility of usingphytosphingosine or its salts as a slimming agent. It has been possibleto confirm this effect.

Hence, according to a first aspect, the present invention relates to anovel cosmetic use of phytosphingosine or of one of its cosmeticallyacceptable salts, particularly its hydrochloride, as a slimming agentfor preparing a cosmetic composition intended for reducing subcutaneousexcess fat.

According to a second aspect, the invention relates to a novel cosmeticuse of phytosphingosine or of one of its cosmetically acceptable salts,particularly of its hydrochloride, as an active agent which stimulatesthe synthesis of leptin by adipocytes, for preparing a cosmeticcomposition intended for reducing subcutaneous excess fat.

According to a third aspect, the invention relates to a method ofcosmetic treatment intended for obtaining a slimming effect on the humanbody, according to which an effective amount of a cosmetic compositioncontaining phytosphingosine or one of its cosmetically acceptable salts,particularly its hydrochloride, is applied on the parts of the bodywhere said effect is sought.

Furthermore, according to the three aspects of the invention, as definedsupra, it has appeared that certain combinations of phytosphingosine orof one of its cosmetically acceptable salts, particularly of itshydrochloride, turned out to be particularly interesting for improvingthe slimming effect obtained by the application of any one of thecompositions containing these particular combinations.

More specifically, it has appeared that the combination ofphytosphingosine or of one of its cosmetically acceptable salts, withone or more agents, hereinafter designated as lipolytic agents, whichinduce a lipolysis, in the adipocytes, turned out to be particularlyinteresting within the context of the present invention, as will beexplained further on.

In particular, at least one cosmetically acceptable lipolytic agent willbe selected from the group consisting of adenosine 3′,5′-cyclicmonophosphate (CAMP) and its derivatives, adenylate cyclase enzymeactivating agents and phosphodiesterase enzyme inhibiting agents, formaking this combination.

Forskolin, or a plant extract containing it, such as an extract ofColeus forskohlii or Plectranthus barbatus, or even an extract of theplant Tephrosia purpurea, will advantageously be selected as adenylatecyclase activating agent.

It will be possible to use a xanthine, such as3-isobutyl-1-methyl-xanthine or IBMX, caffeine or theophilline, asphosphodiesterase inhibiting agent.

The cosmetic compositions containing such combinations, which are novelper se, constitute the fourth aspect of the invention. It is thesecompositions as they are defined infra which will preferably be made useof in all the cosmetic applications covered by the present invention.

Hence, according to this fourth aspect, the present invention relates toa cosmetic composition, notably intended for reducing subcutaneousexcess fat, characterised in that it contains, as active agent,

-   -   phytosphingosine, or one of its cosmetically acceptable salts,        particularly its hydrochloride, and    -   at least one cosmetically acceptable lipolytic agent selected        from the group consisting of CAMP and its cosmetically        acceptable derivatives, adenylate cyclase enzyme activating        agents and phosphodiesterase enzyme inhibiting agents,        in a cosmetically acceptable vehicle.

In the novel compositions of the invention, which are also thecompositions which are preferred for the implementation of the variousapplications covered by the present invention, phytosphingosine, or oneof its cosmetically acceptable salts, particularly its hydrochloride, iscontained in the cosmetic composition at a concentration of between0.001% and 1% and, preferably, between 0.05% and 0.5% by weight withrespect to the total weight of said composition.

The cosmetic composition further contains at least one lipolytic activeagent selected from the group consisting of CAMP and its lipolyticderivatives, adenylate cyclase enzyme activating agents andphosphodiesterase enzyme inhibiting agents.

In these cosmetic compositions, CAMP or its derivative willadvantageously be used at a concentration of between 0.001% and 5% byweight with respect to the total weight of the composition.

It will be possible to select any cosmetically acceptable derivative ofCAMP, and particularly a salt or an acylated derivative, notably a mono-or dibutyryl derivative, as a derivative of CAMP.

Forskolin, or a plant extract containing it, preferably at aconcentration of between 0.001% and 1% and, preferably between 0.05% and0.25%, by weight with respect to the total weight of the composition, isadvantageously selected as an adenylate cyclase enzyme activating agent.

An extract of Coleus forskohlii or Plectranthus barbatus will bepreferably be selected as an extract containing forskolin. Such anextract can be obtained by an extraction method, such as the onedescribed in the International application WO 91/02516.

It will also be possible to use an extract of the plant Tephrosiapurpurea, at a concentration of between 0.001% and 5%, preferablybetween 0.01% and 5%, by weight with respect to the total weight of thecomposition, as an adenylate cyclase activating agent. Such an extractcan be obtained by an extraction method such as the one described in theInternational application WO 95/03780.

Finally, as set forth supra, according to another variant, the preferredcompositions according to the invention contain a phosphodiesteraseinhibiting agent, particularly a xanthine, and, more particularly,3-isobutyl-1-methyl-xanthine (IBMX), caffeine or theophilline,preferably at a concentration of between 0.001% and 10%, preferablybetween 0.01 and 1%, by weight with respect to the weight of thecomposition.

The preferred compositions which are used in accordance with the presentinvention and which contain a combination of phytosphingosine or of oneof its salts with a lipolytic agent such as CAMP and its lipolyticderivatives, adenylate cyclase activating agents and phosphodiesteraseinhibiting agents, turn out to be particularly interesting by virtue ofthe synergistic action of the two types of constituents.

Without the inventors considering to be totally bound by thisexplanation, a plausible interpretation of the synergy effect observedis given infra.

It has in fact already been observed that the agents which promote alipolysis in the adipocytes, such as the extracts of Coleus for example,possess a remarkable biological effectiveness which in general combinesa significant lipolytic power with an inhibitory activity of adipocytematuration. The significant reduction in volume and in quantity of thelipid vacuoles after a treatment with the lipolytic agent leads to areduction in the production of leptin. It is thus probable that thislocal loss of leptin in the environment close to the adipocytesresulting from the treatment by the lipolytic agent might be compensatedby the effect of a product which stimulates leptin synthesis, in thepresent case by phytosphingosine or its salt. The maintenance of asufficient leptin concentration in the environment close to theadipocytes thus exerts a role with acts against the increase in theadipose mass. It thus seems that all takes place as though the messagewere emitted by fatty cells which inform, by a retro-control operation,of the necessity to reduce the storage in the form of triglycerides.Thus, by virtue of the combined action of phytosphingosine or of one ofits salts, and of at least one other lipolytic agent, an increased and alonger-acting slimming effect is obtained.

This hypothesis does seem to be confirmed entirely by the resultsobtained within the context of Examples 2 and 4 of the present inventionwhich concern the combination of phytosphingosine with an extract ofColeus forskohlii.

The following Examples are given purely as an illustration of thepresent invention. They are accompanied by FIGS. 1 to 6, whichrepresent, respectively:

FIG. 1: the effect upon the lipogenesis of various treatments carriedout in Example 2;

FIGS. 2 to 4: the morphology of the murine adipocytes at differentstages of the treatment according to Example 2;

FIG. 5: the morphology of the human adipocytes at D18 for a controlsolution (according to Example 3); and

FIG. 6: the morphology of the human adipocytes at D18 for a treatmentwith a combination according to the invention (according to Example 3).

EXAMPLES

In the following Examples, and, unless indicated otherwise, theproportions are indicated in percentage by weight.

Example 1

Demonstration of the Stimulating Activity of Phytosphingosine on theProduction of Leptin by Murine Adipocytes in Culture.

1. Principle of the Test

The concept according to which the adipose mass may be regulated viasecreted circulating factors is very interesting.

The principle of the test is to control the secretion of leptin by theadipocytes.

2. Material and Methods

Culture of 3T3 F442A Cells

A clone, which has the capacity to accumulate larges amounts oftriglycerides, was isolated from an established cell-line of mouse 3T3preadipocytes. The lipolytic agents reduce this accumulation. It thusappeared important to test potential lipolytic agents on this 3T3 F442Amurine peradipocytes cell-line. These preadipocytes (GREEN H. andKEHINDE O.—Spontaneous Heritable Changes Leading to Increased AdiposeConversion in 3T3 Cells, Cell Vol. 7, 105-113, 1976) can multiply anddifferentiate by possessing the morphological and biochemical phenotypewhich is characteristic of the differentiated function of the matureadipocyte. When they are in exponential growth phase, they are offibroblastic appearance, having an elongated shape and are very adherentto the support. At the confluence, when the conditions so permit, a verypremature morphological transition gives them a rounded shape.

The cells thus undergo a clonal amplification process. Increases in theactivity of lipogenetic enzymes are added to these morphologicalchanges, as well as increases in responses by the cells tohormones/factors which affect the lipogenesis and the lipolysis.

The 3T3 F442A preadipoytes thus constitute an excellent model for studyof lipolysis, by virtue of the morphological and metabolictransformations acquired by the cells during their developmentprogramme. (Pairault J and Lasnier F: Control of adipogeneticdifferenciation of 3T3 F442A cells by retinoic acid, dexamethasone andinsulin: a topographic analysis J. cell Physiol. 1987, 132, 279-86).

According to the literature, leptin is secreted in the culture mediumsince it is stored in the adipocytes. (Wabitsch M et al, Diabetes, 1996,vol 45, Bornstein S. et al, Diabetes, 2000, vol 49, Friedman J M,Nutrition Reviews, 1998, vol 56 no2).

In 3T3 F442A cells, the expression of the ob gene has been studied inparticular (Leroy P et al, J. of Biol. Chem, 1996, vol 271 no5, pp.2365-2368, Considine R V et al—Horm. Res. 1996, 46: 249-256).

The 3T3 F442A preadipocytes are sown at DO in 35 mm Petri dishes(Corning) and placed in an oven at 37° C. under an air-CO₂ atmosphere(95-5). The cells are cultivated in an Eagle minimum essential mediummodified according to Dulbecco (glucose 4.50 g/l) (DMEM-GIBCO BRL)supplemented with 5% of calf serum (CS) (BIOMEDIA®) and 5% of foetalcalf serum (GIBCO) during the growth phase. The medium is changed at D2and D4.

At the cell confluence (at D7), the medium is changed:

The basic medium remains the same (DMEM) but is supplemented with 10% offoetal calf serum (FCS) and insulin (5 μg/mL) (SIGMA).

The medium is then changed at D9 and D11.

At D14, D16 and D18, a treatment is made with the composition theeffectiveness of which on the leptin synthesis it is sought to verify.

The protocol is summarised in the Table below: D0 Sowing of the 3T3F442A in DMEM, 5% CS, 5% FCS - cell density 2 × 10⁴ cells/35 mm Petridish D2 Change of medium D4 Change of medium D7 Confluence - Culturemedium DMEM, 10% FCS, 1% insulin (mother solution at 500 μg/mL) D9 andChange of medium D11 D14 Treatment with the composition to be tested D16and Treatment with the composition to be tested D18 D21 Collection ofthe cell supernatants3. Leptin Determination

The leptin secreted is determined by means of a sandwich-type Elisatechnique re-running with a Quantikine M Mouse Leptin Immunoassay kit.

This ELISA determination uses recombined mouse leptin, expressed in E.coli and antibodies directed against recombinant mouse leptin.

The test uses a “sandwich” immunoenzymatic technique. The microplatewells are lined with a mouse leptin polyclonal antibody.

The standards, controls and samples are deposited in the wells and, atthe same moment, all the leptin present binds to the immobilisedantibody.

The bound leptin is then detected by a mouse anti-leptin antibody whichis coupled to an enzyme peroxidase. A substrate solution is then addedinto the wells. The enzymatic reaction leads to a blue solution whichturns yellow after addition of a quenching solution.

The intensity of the colour measured is proportional to the amount ofleptin present. The reading of the optical density is done at 450 nm onthe spectrophotometer.

The determination is obtained afterwards of the dose-response curves inrelation to the measurement of natural leptin, parallel to the standardcurves obtained with “recombinant” Quantikine M standards. TheQuantikine M kit thus enables the relative mass values for the naturalmouse leptin to be determined.

The optical density measured at 450 nm is proportional to the amount ofantibody fixed, which is itself proportional to the amount of leptinpresent initially. The results are expressed in pg/mL of leptin presentin the cell supernatants.

The samples were tested in triplicate.

4. Results

Three compositions containing 0.25, 1 and 2 μg/mL, respectively, ofphytosphingosine or of its hydrochloride, were tested.

For the 3T3-F442A adipocytes which are maintained in culture without anytreatment, the amount of leptin present in the culture supernatantsincreases strongly with time (16.4 pg/mL at D4, 802 pg/mL at D11 and2,623 pg/mL at D20). This result is in conformity with the biologicaldata (Leroy P. 1996, Considine R V. 1996): under basal conditions, themurine adipocytes secrete increasing amounts of leptin all throughouttheir maturation. We thus confirm that a mature adipocyte secretesamounts of leptin which are greater than those of a preadipocyte.

The amounts of leptin present in the supernatants are reported in Table1 below. The abbreviation PS designates phytosphingosine. TABLE 1 Amountof leptin, expressed in pg/mL, which is present in the culturesupernatants of mature 3T3-F442A adipocytes treated withphytosphingosine at day D21 leptin (pg/mL) Standard phytosphingosine(μg/mL) Average deviation O (control) 1510 23.43 0.25 1789 11.27 11563.6 14.33 2 1428.8 200

After 7 days of treatment, i.e. at day D21, phytosphingosine induces anincrease in the secretion of leptin by treated adipocytes, an effectwhich is maximum at the concentration of 0.25 μg/mL. The increase is ofa little more than 18% at this concentration.

Phytosphingosine hydrochloride, when tested under the same conditions,is also responsible for a stimulation of the leptin secretion: +52% at 1μg/mL and +26% at 2 μg/mL and +18% at 0.25 μg/mL. The results for thehydrochloride are reported in Table 2 below. TABLE 2 Amount of leptin,expressed in pg/mL, which is present in the culture supernatants ofmature 3T3-F442A adipocytes treated with phytosphingosine hydrochlorideat day D21 leptin (pg/mL) phytosphingosine-HCl Standard (μg/mL) Averagedeviation 0 (control) 1510 23.43 0.25 1789 201.79 1 2301.3 49.821 21907.9 31.93

Thus, phytosphingosine is capable of inducing an increase in the basaladipocyte secretion of leptin in the 3T3-F442A adipose cell, a modelwhich is very close to the human adipocyte. Phytosphingosine is thuscapable of playing an important role in the control of the stability ofthe fatty mass.

Example 2

Demonstration of the Interest in the Combination of Phytosphingosinewith an Adenylate Cyclase Activator, Such as an Extract of Coleusforskohlii, for Promoting the Decrease of Lipogenesis in MurineAdipocytes in Culture.

1. Principle of the Study

This study relates to the effects of the combination of the two actives,Coleus forskohlii (also named Plectranthus barbatus) (PB) andphytosphingosine (PS) on the recruiting of new adipocytes.

It is known that the development of white adipose tissue represents aprocess which is continuous throughout the whole life (AILHAUD G.,GRIMALDI P., NEGREL R., Trends in Endocrinology and Metabolism, (1994) 5(3) 132-6). The adipocyte is associated, within the adipose tissue, withan abundant extracellular matrix which also includes endothelial cells,capillaries, nerve fibres and fibroadipoblast precursors. The matureadipocyte represents the phenotype of a cell originating from thedifferentiation of an adipocyte precursor. The preadipocytes are presentwithin the same adipose tissue and can be recruited at any stage of lifein order to generate new adipocytes: in the case of a weight gain, aninitial phase exists of increase in the adipocyte volume until acritical point is attained, which then leads to the recruitment of newcells (Bjorntorp P., Int. J. Obesity, (1991) 15 67-81). The intrinsiccapacity of the preadipocytes to multiply and to differentiate intoadipocytes plays a determinant role in the development of fatty masses.A hyperplasia of these cells related to the fibroblasts leads to anincrease of the adipose tissue.

Thus, the mature adipocytes are firstly treated with PB+PS.

Secondly, the culture medium which is conditioned with these adipocytesis placed in contact with preadipocytes the maturation of which intoadipoctyes will be followed.

The control cells at the start of the treatment commence todifferentiate and undergo a certain number of changes: increase involume, increase in number and of the size of the lipid droplets,increase in the activity of lipogenetic enzymes, etc.

A key enzyme in the process of synthesis of triglycerides isglycerol-3-phosphate dehydrogenase (G₃PDH): its specific activityincreases considerably during maturation and can thus be used as aprecise and sensitive measurement of adipocyte conversion (Pairault J.,Green H., Proc. Nat. Acad. Sci. USA, (1979) 76, 5138-42; Koekemoer T. C.et al, Int. J. Biochem. Cell Biol. (1995) 27, 625-32).

It was chosen to follow the evolution of the activity of this enzyme inorder to measure the state of differentiation of the adipocytes.

Since G₃PDH is a hydrosoluble enzyme, its activity is measured in thesupernatant of the cell grindings, in the presence of appropriatesubstrates (NADH, TEA (triethanolamine)—EDTA, 50 mM, 1 mM).

The specific activity is calculated from these determinations. Thetreated cells are compared with the control cells. Since G₃PDH is areflection of the state of differentiation of the cells, the higher itsspecific activity, the more the cells are differentiated, and viceversa: the more limited the fat reserve will be by the agent tested,more the activity of the G₃PDH will be lower.

The average over three measurements with respect to a standard deviationgives an average specific activity. Then, the percentage inhibition iscalculated of the inhibition of the activity of the G₃PDH produced bythe substances compared to the controls.

The criteria which enable ensuring the quality of good anti-lipogeneticagents are on the one hand a percentage inhibition of the enzyme whichis greater than 50%, and on the other hand crude data which aresignificantly different with respect to the controls.

2. Material and Methods

a) Culture and Treatments

The adipocytes undergoing differentiation, not very mature adipocytes,also named preadipocytes, obtained at D7 of the protocol given inExample 1 are treated with the medium which is conditioned with maturedifferentiated adipocytes which are not treated or treated with PB or PSand with the combination PB+PS for 8 days with a change of medium atdays D9 and D11, as is indicated in the protocol below.

The phytosphingosine was tested at the final concentrations of 0.25,0.50 and 1 μg/mL.

The extract de Plectranthus barbatus (PB) (batch No. 0B2, INDENA) istitrated at 80% of forskolin, a molecule which is recognised for beingan effector of adenylate cyclase (Seamon K. et al., P.N.A.S. USA, (1981)78 3363-67). The PB concentration used in the study is 25 μg/mL from amother solution at 20 mg/mL in ethanol.

b) Preparation of the Cell Extracts

The cell plug is washed twice with PBS buffer and the cells arerecovered by scratching in a 25 mM TRIS—HCl buffer, pH 7.5, containing 1mM of EDTA at 4° C. The cells are homogenised by grinding andcentrifugation at 10,000 g for 10 minutes at 4° C.

The protocol followed is summarised in the Table below. D0 Sowing of the3T3F442A - cell density 2 × 10⁴ cells/35 mm Petri dish. Culture mediumDMEM, 5% FCS/5% CS. D2 Change of medium D4 Change of medium D7Confluence - culture medium DMEM, 10% FCS, 1% insulin (SM 500 μg/mL)Treatment with conditioned media: PB: 0.25 μg/mL PS: 0.5 and 1 μg/mLD9-D11 Change of medium, same medium + treatment D14 Treatment, sameculture medium D16 Collection of the supernatants and G₃PDHdetermination

c) Determination of the glycero-3-phosphate Dehydrogenase (G₃PDH)Activity.

The determination of the G₃PDH activity is made according to the methodof Kozak and Jensen: Kozak and Jensen, 1974, J. Biol. Chem., 249,7775-7781.

G₃PDH catalyses the following reaction

The consumption of NADH as a function of time is measured byspectrophotometry (KONTRON) at 340 nm. An absorbance variation/minute (ΔAbs/minute) can thus be calculated which corresponds to the initial rateof the enzymatic reaction. The results are expressed in specificactivity (SA), i.e. in nmoles of NADH transformed/min/mg of proteins.The total protein content is evaluated by the BCA method (Protein AssayReagent-PIERCE LTD)${SA} = {81.25 \times \Delta\quad{{Abs}/\min} \times \frac{1}{{mg}\quad{of}\quad{proteins}}}$3. Results

Measurement of the glycero-3-phosphate dehydrogenase (G₃PDH) activity.

The amounts of NAD+ after 8 days as a function of the treatments of thecultures are reported in Table 3 below. TABLE 3 Appreciation of thelipogenesis by measurement of the OD, expressed in nmoles NAD+, for thePB extract and for the phytosphingosine PS PB PS O.D. (nmoles NAD+)(μg/mL) (μg/mL) average Standard deviation 0 (control) 0 (control)0.0016 0.000195 25 0 0.0004 0.000015  0 0.5 0.0014 0.00009  0 1 0.001650.00006 25 1 0.00036 0.00002 25 0.5 0.00043 0.000048

The results of Table 3 above are also represented in FIG. 1.

After 8 days' contact with the media which are conditioned with matureadipocytes, a slowing down of the maturation of the peradipocytes withthe media containing PB (inhibition of the G₃PDH activity) is observed,and this corresponds to an inhibition of the recruitment of thepreadipocytes, the activity of which of the maturation marker enzyme isinhibited by 75%.

Phytosphingosine does not modify this anti-lipogenetic profile the PS+PBcombination causes between 74 and 79% of inhibition of the G₃PDHactivity, the 1 μg/mL PS+25 μg/mL PB combination even leads to a slowingdown of the lipogenesis of these preadipocytes during maturation whichis slightly greater than with PB alone.

b) Analysis of the Morphology of the Adipocytes

In parallel, the morphology of the adipocytes was analysed by directobservation of the cells in the Petri dishes, under reverse phasemicroscope (Olympus BH2). The cells are considered to be differentiatedby morphological analysis when they acquire a round surround and thattheir cytoplasm is filled with lipid droplets. Inversely, a decrease inthe amount of lipid vacuoles which is associated with a more elongatedform gives evidence of a slowing down of this maturation.

In the presence of the PS+PB combination, the cells are characterised bya very marked delipidation of the adipocytes, which is in agreement withthe lowering of the activity of the G₃PDH enzyme measured before.

FIGS. 2, 3 and 4 show, respectively:

FIG. 2: the cells of a control culture at D7. In this culture, the cellsare not very differentiated, there are not many lipid vacuoles. This isthe start of the treatment.

FIG. 3: the cells of a control culture at D21. The cells are loaded withlipid vacuoles.

FIG. 4: the cells of a culture treated with the 25 μg/mL PB+1 μg/mL PScombination.

c) CONCLUSION

This study shows that the treatment with the PB+PS combination of matureadipocytes gives information which is capable of all in all slowing downthe accumulation of triglycerides in peradipocytes. The lowering of theactivity of the lipogenetic enzyme G₃PDH leads to a depletion insignificant intracellular lipid droplets.

The addition of phytosphingosine capable of stimulating the secretion ofleptin did not inhibit the massive action of the PB on the decrease inlipids.

The maintenance of leptin in the environment close to the adipocytescould thus exert its role of signal molecule acting against the increasein the adipose mass. One is tempted to believe that a collection ofcells containing cells which are emptied of a part of their content oftriglycerides by lipolysis in continuing to emit a retrocontrol leptinmessage should contribute to the reduction of the storage of fat by thepanniculus adiposus.

Example 3

Demonstration on Human Adipocytes in Culture of the Activity ofPhytosphingosine and of the Combination of Phytosphingosine with anExtract of Coleus forskohlii, on the Production of Leptin and onLipolysis.

Human adipocytes originating from a plasty of a 41 year old woman, whichare marketed by ZEN-BIO (USA), were used at an advanced stage of theadipocyte maturation. The reception of these cells in culture took place16 days after sowing. They are cultivated in a specific medium ensuringtheir differentiation throughout the whole treatment.

-   D-16: sowing-   D0: start of the treatment with the extract of Coleus (PB) and/or    phytosphingosine (PS)-   D6 to D18: determination of the various parameters.

The properties of Coleus described above for the 3T3F442A murineadipocytes were first of all verified on these human cells: lipolyticactivity by measurement of the release of glycerol and non-esterifiedfatty acids.

Table 4 below indicates the values of glycerol release in the controlcultures and cultures treated with PS. TABLE 4 Release of glycerol,expressed in μg/mL, by human adipocytes in culture, which arenon-treated or treated with 25 μg/mL of phytosphingosine, as a functionof the duration of the culture glycerol (pg/mL) Days Control (PS = 0μg/mL) PS = 25 μg/mL 3 73.78 174.4 6 120.8 196.2 9 147.2 302.9 12 198.4416.4 15 196.3 460.6 18 230.9 659.8 21 276.6 777.1 23 238.9 687

It appears very clearly that the Coleus very strongly stimulates theadipocyte lipolysis (+128% increase in release of glycerol at 18 dayswith respect to the control).

It is further observed, by a measurement of release of non-esterifiedfatty acids, that the Coleus causes a strong hydrolysis of the adipocytetriglycerides in releasing, in parallel to the glycerol, a large amountof non-esterified fatty acids, at a dose of 25 μg/mL.

Other tests show that the phytosphingosine induces an increase of leptinsecretion in human adipocytes as in murine adipocytes, the mosteffective doses being lower for the human adipocyte.

In parallel to the leptin release, phytosphingosine, at the dose of 6ng/mL is responsible for a release of glycerol with time, withoutconcomitant release of non-esterified fatty acids. This observationcould correspond to a new form of lipolysis which is proper to leptin,and which is already described in publications.

FIGS. 5 and 6 present the evolution of the morphology of the cells underthe conditions which have just been set forth.

FIG. 5 presents a control culture of human adipocytes at day D18.

A significant amount of fat vacuoles is clearly seen.

FIG. 6 presents a culture of human adipocytes at day D18, aftertreatment by the combination of Coleus (25 μg/mL) with phytosphingosine(6 ng/mL). It appears clearly that the phytosphingosine-Coleuscombination leads to a massive and visible decrease, with lipid dropletswhich are less numerous and of smaller size. In parallel, a highernumber of cells take up an appearance of not very mature cells(elongated form and absence of lipid vacuoles).

Thus, the combination of these two actives leads to a powerful reductionin the fat store, the modulation of leptin in the environment close tothe human adipocyte being a key step in this action.

Example 4

Slimming hydro/alcoholic formulation. Water qsp 100% Denatured alcohol  42% PPG-3 myristyl ether   10% Perfume 0.20% Phytosphingosine 0.10%

Example 5

Slimming fluid emulsion. PPG-2 isoceteth-20 acetate   2% Poloxamer 4070.50% Propylene glycol isoceteth-3 acetate   15% Pentacyclomethicone  15% Water qsp.100% Butylene glycol   3% Preservatives q.s. Extract ofColeus forskohlii (at 80% of forskolin)  0.1% Xanthan gum 0.05%Acrylates/c10-30 alkyl acrylate cross-polymer 0.04% Neutraliser q.s.Polyacrylamide c13-14 isoparaffin laureth-7 0.50% Perfume 0.20%Phytosphingosine 0.05%

Example 6

Slimming cream. Steareth-2  0.50% Steareth-21  1.75% Cetyl alcohol 0.30% Stearyl alcohol  0.30% Stearic acid  0.50% 2-ethylhexyl stearate 4.00% Cetearyl isononanoate  3.00% Squalane  4.00% IBMX  1.00%Dimethicone  0.40% Water qsp.100.00% Glycerine  2.00% Butylene glycol 3.00% Preservatives q.s. Acrylates/c10-30 alkyl acrylate cross-polymer 0.35% Xanthan gum  0.10% Sodium hyaluronate  0.02% Neutraliser q.s.Polyacrylamide c13-14 isoparaffin laureth 7  0.50% Denatured alcohol 5.00% Perfume  0.20% Phytosphingosine 0.002%

1-30. (canceled)
 31. A method of cosmetic treatment for obtaining aslimming effect on the human body, comprising the application on theparts of the body in need thereof of an effective amount of a cosmeticcomposition containing an active agent selected from the groupconsisting of phytosphingosine and its cosmetically acceptable salts.32. The method according to claim 31, wherein said method is intendedfor reducing subcutaneous excess fat.
 33. The method according to claim31, wherein said active agent is phytosphingosine hydrochloride.
 34. Themethod according to claim 31, wherein the concentration of said activeagent is comprised between 0.001% and 1%, by weight with respect to thetotal weight of said composition.
 35. The method according to claim 31,wherein said composition further comprises at least one cosmeticallyacceptable lipolytic agent.
 36. The method according to claim 35,wherein said lipolytic agent is selected from the group consisting ofadenosine-3′,5′-cyclic monophosphate (CAMP) and its cosmeticallyacceptable derivatives.
 37. The method according to claim 36, whereinsaid cosmetically acceptable derivatives are selected from the groupconsisting of the salts and acylated derivatives of CAMP.
 38. The methodaccording to claim 37, wherein said derivative is selected from thegroup consisting of mono- and dibutyril derivatives of CAMP.
 39. Themethod according to claim 36, wherein said CAMP or said lipolytic agentis at a concentration of between 0.001% and 5%, by weight with respectto the total weight of said composition.
 40. The method according toclaim 35, wherein said lipolytic agent is an adenylate cyclase enzymeactivating agent.
 41. The use according to claim 40, wherein saidadenylate cyclase enzyme activating agent is selected from the groupconsisting of forskolin and plant extracts containing the same.
 42. Themethod according to claim 41, wherein said adenylate cyclase enzymeactivating agent is at a concentration of between 0.001% and 1%, byweight with respect to the total weight of said composition.
 43. Themethod according to claim 41, wherein said adenylate cyclase enzymeactivating agent is selected from the group consisting of extracts ofColeus forskohlii and Plectranthus barbatus.
 44. The method according toclaim 41, wherein said adenylate cyclase activating agent is an extractof the plant Tephrosia purpurea, at a concentration of between 0.001%and 5% by weight, with respect to the total weight of said composition.45. The method according to claim 35, wherein said lipolytic agent is aphosphodiesterase enzyme inhibiting agent.
 46. The method according toclaim 45, wherein said phosphodiesterase enzyme inhibiting agent isselected from the group consisting of xanthines, IBMX, caffeine andtheophylline.
 47. The method according to claim 46, wherein saidphosphodiesterase enzyme inhibiting agent is3-isobutyl-1-methyl-xanthine.
 48. The method according to claim 46,wherein said phosphodiesterase enzyme inhibiting agent is at aconcentration of between 0.001% and 10% by weight, with respect to theweight of said composition.
 49. A method of cosmetic treatment forstimulating the synthesis of leptin by adipocytes, comprising theapplication on the parts of the body in need thereof of an effectiveamount of a cosmetic composition containing an active agent selectedfrom the group consisting of phytosphingosine and its cosmeticallyacceptable salts.
 50. The method according to claim 49, wherein saidmethod is intended for reducing subcutaneous excess fat.
 51. The methodaccording to claim 49, wherein said active agent is phytosphingosinehydrochloride.
 52. The method according to claim 49, wherein theconcentration of said active agent is comprised between 0.001% and 1%,by weight with respect to the total weight of said composition.
 53. Themethod according to claim 49, wherein said composition further comprisesat least one cosmetically acceptable lipolytic agent.
 54. The methodaccording to claim 53, wherein said lipolytic agent is selected from thegroup consisting of adenosine-3′,5′-cyclic monophosphate (CAMP) and itscosmetically acceptable derivatives.
 55. The method according to claim54, wherein said cosmetically acceptable derivatives are selected fromthe group consisting of the salts and acylated derivatives of CAMP. 56.The method according to claim 55, wherein said derivative is selectedfrom the group consisting of mono- and dibutyril derivatives of CAMP.57. The method according to claim 56, wherein said CAMP or saidlipolytic agent is at a concentration of between 0.001% and 5%, byweight with respect to the total weight of said composition.
 58. Themethod according to claim 53, wherein said lipolytic agent is anadenylate cyclase enzyme activating agent.
 59. The use according toclaim 58, wherein said adenylate cyclase enzyme activating agent isselected from the group consisting of forskolin and plant extractscontaining the same.
 60. The method according to claim 59, wherein saidadenylate cyclase enzyme activating agent is at a concentration ofbetween 0.001% and 1%, by weight with respect to the total weight ofsaid composition.
 61. The method according to claim 59, wherein saidadenylate cyclase enzyme activating agent is selected from the groupconsisting of extracts of Coleus forskohlii and Plectranthus barbatus.62. The method according to claim 59, wherein said adenylate cyclaseactivating agent is an extract of the plant Tephrosia purpurea, at aconcentration of between 0.001% and 5% by weight, with respect to thetotal weight of said composition.
 63. The method according to claim 53,wherein said lipolytic agent is a phosphodiesterase enzyme inhibitingagent.
 64. The method according to claim 63, wherein saidphosphodiesterase enzyme inhibiting agent is selected from the groupconsisting of xanthines, IBMX, caffeine and theophylline.
 65. The methodaccording to claim 64, wherein said phosphodiesterase enzyme inhibitingagent is 3-isobutyl-1-methyl-xanthine.
 66. The method according to claim64, wherein said phosphodiesterase enzyme inhibiting agent is at aconcentration of between 0.001% and 10% by weight, with respect to theweight of said composition.
 67. A cosmetic composition, notably intendedfor reducing subcutaneous excess fat, containing, as active agents:phytosphingosine or one of its cosmetically acceptable salts, and atleast one lipolytic agent selected from the group consisting of CAMP andits cosmetically acceptable lipolytic derivatives, adenylate cyclaseenzyme activating agents and phosphodiesterase enzyme inhibiting agents,in a cosmetically acceptable vehicle.
 68. The composition according toclaim 67, wherein said cosmetically acceptable salt is phytosphingosinehydrochloride.
 69. The cosmetic composition according to claim 67,containing from 0.001 to 1%, by weight of phytosphingosine or of one ofits cosmetically acceptable salts.
 70. The cosmetic compositionaccording to claim 67, wherein said lipolytic agent is selected from thegroup consisting of CAMP and its cosmetically acceptable derivatives.71. The cosmetic composition according to claim 70, wherein saidcosmetically acceptable derivative of CAMP is selected from the groupconsisting of the salts and acylated derivatives of CAMP.
 72. Thecosmetic composition according to claim 71, wherein said cosmeticallyacceptable derivative of CAMP is selected from the group consisting ofmono- and dibutyryl derivatives of CAMP.
 73. The cosmetic compositionaccording to claim 70, wherein said lipolytic agent is at aconcentration of between 0.001% and 5% by weight with respect to thetotal weight of said composition.
 74. The cosmetic composition accordingto claim 67, wherein said the lipolytic agent is an adenylate cyclaseenzyme activating agent.
 75. The cosmetic composition according to claim74, wherein said adenylate cyclase enzyme activating agent is selectedfrom the group consisting of forskolin and plant extracts containing thesame.
 76. The cosmetic composition according to claim 75, wherein saidadenylate cyclase enzyme activating agent is at a concentration ofbetween 0.001% and 1%, by weight with respect to the total weight ofsaid composition.
 77. The cosmetic composition according to claim 75,wherein said adenylate cyclase enzyme activating agent is an extract ofColeus forskohlii or of Plectranthus barbatus.
 78. The cosmeticcomposition according to claim 74, wherein said adenylate cyclaseactivating agent is an extract of the plant Tephrosia purpurea.
 79. Thecosmetic composition according to claim 78, wherein the extract ofTephrosia purpurea is at a concentration of between 0.001% and 5% byweight, with respect to the total weight of the composition.
 80. Thecosmetic composition according to claim 67, wherein said lipolytic agentis a phosphodiesterase enzyme inhibiting agent.
 81. The cosmeticcomposition according to claim 80, wherein said phosphodiesteraseinhibiting enzyme agent is selected from the group consisting ofxanthines, caffeine and theophylline.
 82. The cosmetic compositionaccording to claim 81, wherein said phosphodiesterase inhibiting enzymeagent is selected from the group consisting of3-isobutyl-1-methyl-xanthine and IBMX.
 83. The cosmetic compositionaccording to claim 81, wherein said phosphodiesterase inhibiting enzymeagent is at a concentration of between 0.001% and 10%, by weight withrespect to the total weight of the composition.